Mold culture



proteolytic Patented Mar. 31, 1942 UNI-TED STATES PATENT. OFFICE MOLD CULTURE Gilbert B. Ayres, Stamford, and Joseph George Niedercorn,

Riverside, Com, American Cyanamid Company,

assignors to New York,

N. Y., a corporation of Maine Application June 16, 1939, Serial No. 279,472

1 Claim.

tractive in the bating of hides and in similar processes involving the selective digestion of proteins.

It is an object of the present invention to provide these enzymes in the form of a dried proteolytic culture which is suitable for use as a bate. It is a further object to provide a method.

for the bating of hides and skins using these enzymes as bates, preferably in conjunction with a deliming agent such as ammonium sulfate;

The invention includes both the production of these enzymes from Penicillium brevicaule by inoculation of a suitable nutrient medium and cultivation of the inoculated medium and the provision of the enzymes and nutrient medium or carrier in dried form, and also the selective digestion of proteins and the hating of hides and skins by subjecting the materials to the action of these proteolytic enzymes. I

As illustrative of the proteolytic activity in alkaline solution of these enzymes produced by Penicillium brevicaule there is shown in the single figure of the accompanying drawing a curve, the ordinates of which are given in terms of standard alkali and the abscissae in terms of pH values. The curve was constructed from values obtainedin digesting a casein substrate (sodium caseinate) with a culture of these enzymes at increasing alkalinities of the sodium caseinate' solution. The undigested casein was precipitated with a standardized solution of sulfuric acid and sodium sulfate and the filtrate therefrom, containing the digested casein, ti-

trated with 0.1 N-alkali. The tests were conducted by a modification of the procedure of Volhard and Loehlein for the determination of activity of enzymes described in Praktikum der Physiologischen Chemie, part 1, pages 258-259, by Peter Rona, 2d ed., Julius Springer, Berlin, 1931. The curve which was plotted for a pI-lrange of 7-10, after sloping off from the point of neutrality, changes sign and from this curve that the enzymes of the present invention manifest a uniformly good rate of activity in' alkalies at least up to the point where the alkalinity reaches a pH value of about 9.5.

The preparation of these enzymes is as follows: A suitable nutrient medium in the granular or solid discrete particle condition, such as bran, moistened with an equal weight of water, is inoculated with a culture of Penicillium brevicaule and the inoculated moist bran spreadout in thin layers on trays. The inoculated bran is then incubated in an oven maintained at a temperature of about 30 C. and preferably at not higher than this temperature, and at a humidity in the oven such that the atmosphere therein is saturated but does not contain sufiicient moisture to cause deposition of the same onto the bran. The inoculated bran is maintained in the oven until sporulation occurs. 'After incubation for the optimum period the bran culture may be thoroughly mixed with 0.2% of cresylic acid in solution, if desired, to improve the wetting out of the bran when used in solution. The culture is then dried at a temperature below 45 C., e. g. 40 C., and may be used as such for hating, or an ammonium salt, e. g. ammonium sulfate, may be incorporated with the moist mass and the mixture then dried. The bran used for the culture may or maynot be sterilized before the inoculation and likewise the culture may or may not be sterilized, although sterilization of the culture does not appear necessary at the present time.

The enzyme may be liberated from the dried culture by elution with dilute solutions of various salts, such as ammonium chloride, ammonium sulfate, sodium chloride, sodium sulfate, etc. and the eluted enzymes used in other fields as digestants.

The proteolytic enzymes may be applied to the hating of hides-and skins in the form of the combined enzymes and nutrient medium or carrier in any manner now practiced in the art for the application of other tryptic bates. method now in use involves washing the hides from the dehairing step, and adding an ammonium salt, such as ammonium sulfate, to the water containing the hides in order to lower salt the hidesmay be treated for removing lime ascends until a pH of about 9.5 is reached where- Y blast by the addition of a suitable acid, such as hydrochloric acid, to the water bath containing the hides. After the pH of the bath has been suitably adjusted, the enzyme bate is added One thereto in an amount determined by the enzyme unit strength of the bate, th kind of hide or skin to be bated, the extent of hating desired, the length of the bating time and the temperature of the bating bath.

For the purpose of illustration, there is described in the following example a method for bating of hides with the enzymes of Penicillium brevicaule.

Example ture prepared as described above. Run the paddle for the desired length of bating time with a final temperature therein of 85 After the kips have been bated cool them to 70 F.

While the application of these enzymes has been described with particular reference to the bating of hides and skins their utility is not limited to this field. On the contrary they may be used iri many other processes in which a tryptic enzyme of high activity is desired, such .as in desizing and degumming textiles, paper sizing, tenderizing of meat, stripping of gelatin from photographic films and plates, manufacture of peptones, chewing gum, glue, foods, drugs and biological products or in any field where by their use a protein or a protein degradation product can be reduced to a lower molecular size of increased solubility.

It will be understood that the above description is intended as illustrative and not as limiting of the invention, the scope of which is defined by the following claim.

tions of heat and moisture whereby the bran is impregnated with enzymes of proteolytic activity, and drying the incubated culture.

' GILBERT B. AYRES.

JOSEPH G. NIEDERCORN. 

